Ultrasmall cationic superparamagnetic iron oxide nanoparticles as nontoxic and efficient MRI contrast agent and magnetic-targeting tool

Abstract

Fully dispersible, cationic ultrasmall (7 nm diameter) superparamagnetic iron oxide nanoparticles, exhibiting high relaxivity (178 mM(-1)s(-1) in 0.47 T) and no acute or subchronic toxicity in Wistar rats, were studied and their suitability as contrast agents for magnetic resonance imaging and material for development of new diagnostic and treatment tools demonstrated. After intravenous injection (10 mg/kg body weight), they circulated throughout the vascular system causing no microhemorrhage or thrombus, neither inflammatory processes at the mesentery vascular bed and hepatic sinusoids (leukocyte rolling, adhesion, or migration as evaluated by intravital microscopy), but having been spontaneously concentrated in the liver, spleen, and kidneys, they caused strong negative contrast. The nanoparticles are cleared from kidneys and bladder in few days, whereas the complete elimination from liver and spleen occurred only after 4 weeks. Ex vivo studies demonstrated that cationic ultrasmall superparamagnetic iron oxide nanoparticles caused no effects on hepatic and renal enzymes dosage as well as on leukocyte count. In addition, they were readily concentrated in rat thigh by a magnet showing its potential as magnetically targeted carriers of therapeutic and diagnostic agents. Summarizing, cationic ultrasmall superparamagnetic iron oxide nanoparticles are nontoxic and efficient magnetic resonance imaging contrast agents useful as platform for the development of new materials for application in theranostics.

Keywords: MRI; cationic USPIOs; contrast agent; in vivo toxicity; intra-vital microscopy; magnetic targeting.

Figure 1

Transmission electron microscopy image of cat-USPIOs functionalized with aminoethylphosphate (A), the corresponding size distribution histogram (B), and high-resolution TEM image showing the characteristic (220) interplanar distances in a magnetite nanocrystal (C) The arrows indicates the interplanar distance of 0.29 nm in the magnetite 220 crystal plane. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscopy.

Figure 2

Renal clearance after cat-USPIOs IV administration in male Wistar rat. Notes: (A) Typical series of MRI images of a Wistar rat after IV administration of 50 mg/kg of cat-USPIOs, highlighting the variation in the density of white pixels in the kidneys, indicated by yellow circles. The leftmost image was taken before injection of cat-USPIOs (control), and then after 2, 4, and 24 hours after injection. The head of the rat is pointing up. (B) The average of normalized density of white pixels of four subsequent scans of kidneys as a function of time after IV administration of 50 mg/kg of cat-USPIOs. The concentration is inversely proportional to the density of white pixels in magnetic resonance signals. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; MRI, magnetic resonance imaging; IV, intravenous.

Figure 3

Typical series of MRI images of a Wistar rat treated with 50 mg/kg of cat-USPIOs as a function of time from zero up to 140 days after IV administration, highlighting the contrast in liver and spleen. Notes: The negative contrast in the left thigh is due to the accumulation of cat-USPIOs induced by a magnet placed on that local for 2 hours after IV injection. The liver and the spleen are indicated by a yellow rectangle; and a blue circle indicates the negative contrast in the thigh. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; MRI, magnetic resonance imaging; IV, intravenous.

Figure 4

Typical series of MRI images of a Wistar rat treated with 50 mg/kg of cat-USPIOs as a function of time from zero up to 140 days after IV injection, confirming the magnetically induced accumulation of cat-USPIOs in the left thigh by a magnet placed in the local during 2 hours (blue circle). Note: The negative contrast shown in the right thigh (yellow circle) is due to the subsequent intramuscular administration of 15 mg/kg of cat-USPIOs. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; MRI, magnetic resonance imaging; IV, intravenous.

Figure 5

Number of leukocytes (A), lymphocytes (B), and neutrophils (C) present in 1 mm³ of blood before IV administration (time zero), and as a function of time up to 28 days after IV administration of 10 mg/kg of cat-USPIOs in Wistar rats (n=3). Notes: The controls are saline (n=3) and vehicle solution (n=3) (10 mM acetate buffer pH 4.5). No significant statistical difference was observed among the groups. Abbreviations: IV, intravenous; cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles.

Figure 6

Kinetic follow-up of blood dosage of hepatic enzymes (A) ALT, (B) AST, (C) urea, and (D) urine and (E) plasma creatinine from Wistar rats treated with 10 mg/kg of cat-USPIOs as a function of time up to 28 days after IV injection. Notes: A magnet was placed (experimental) or not (control) in the left thigh of a group of rats (n=3) for 2 hours. The controls are saline (n=3) and vehicle solution (n=3) (10 mM acetate buffer pH 4.5). *P<0.05 and **P<0.001 versus respective saline treatment. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; IV, intravenous.

Figure 7

Iron content ([Fe] µg·L⁻¹) determined by GFAAS in the urine of rats (n=3) as a function of time after intravenous injection of 10 mg/kg of cat-USPIOs. The experimental group (n=3, blue) has a magnet placed on the left leg thigh for 2 hours in order to concentrate the magnetite nanoparticles, while the control group (green) did not. Abbreviations: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles; GFAAS, graphite furnace atomic absorption spectrometer.

Figure 8

Effect of cat-USPIOs on leukocyte-endothelial interactions evaluated in male Wistar rat liver sinusoids by intravital microscopy. Notes: (A) Fluorescence intravital microscopy images showing the number of rhodamine 6G-labeled leukocytes adhered to the liver sinusoids 3 hours after intravenous injection (1 mL, single dose) of saline (control, above) and 10 mg/kg cat-USPIOs suspension (experimental, below). (B) Average number of adhered leukocytes in 100 µm segments of sinusoid walls considering at least five sinusoids per rat. White arrows indicate adhered leukocytes. Bar =100 µm. Abbreviation: cat-USPIOs, cationic ultrasmall superparamagnetic iron oxide nanoparticles.